Influenza virus routinely kills over 30,000 Americans per year and has the potential to cause a pandemic that could kill millions and cause severe economic disruption. Although anti-viral drugs exist, these give rise to resistant viruses fairly readily. A better understanding of influenza virus biology has the potential to suggest novel treatment strategies that the virus would find difficult to circumvent. This grant application is prompted by our recent, unexpected discovery that influenza virus expresses high levels of small, ~20 nt long non-coding RNAs in infected cells. These RNAs are not random genome fragments, but instead derive almost entirely from the 5'end of the viral genomic RNA (vRNA) segments. We hypothesize that these RNAs play a key role in the viral life cycle by disrupting cellular innate antiviral immune responses and/or by regulating the transition from mRNA to vRNA and complementary RNA transcription late in the viral life cycle. We intend to fully characterize the origin, temporal expression and sequence identity of these small viral RNAs and to then rigorously test whether blocking these small RNAs, by introducing antisense locked nucleic acid (LNA) and/or antagomir oligonucleotides, will inhibit one or more key steps in the viral life cycle. Conversely, we will also test the effect of overexpressing these small RNAs early in the viral life cycle by transfecting synthetic RNA molecules. Importantly, an inhaled antisense drug that targets another respiratory virus, RSV, is currently in clinical trials, and it is likely that an oligonucleotide that inhibits influenza virus replication effectively could also be delivered by inhalation. Moreover, because the 13-nt sequence found at the 5'end of all influenza virus vRNAs is invariant, it is likely that an effective antisense drug targeted to this region would not select for resistance mutations. In conclusion, we present initial data demonstrating, for the first time, that influenza virus expresses a high level of a specific population of small viral RNAs in infected cells. We seek funds to further characterize the identity and functions of these small viral RNAs and, most importantly, to analyze their potential as targets for antiviral drugs. PUBLIC HEALTH RELEVANCE: We have recently discovered that influenza virus expresses very high levels of small RNA molecules in infected cells. We seek to define the role of these RNAs in the viral life cycle and to evaluate their potential as novel antiviral drug targets.